Sains Malaysiana 40(10)(2011): 1087–1095

Genetic Characterization of Two Mahseer Species (Tor douronensis and Tor tambroides) Using Microsatellite Markers from Other Cyprinids

(Pencirian Genetik dua Spesies Mahseer (Tor douronensis dan Tor tambroides) Menggunakan Penanda Mikrosatelit daripada Siprinid yang Berbeza)

YUZINE B. ESA, SITI SHAPOR SIRAJ*, KHAIRUL ADHA A. RAHIM, SITI KHALIJAH DAUD, HO GHIM CHONG & TAN SOON GUAN, MUHAMMAD FADHIL SYUKRI

ABSTRACT

This study examined the genetic characteristics of twenty-six microsatellite primers developed from three cyprinid fishes (Cyprinus carpio Linnaeus, Barbus barbus Linnaeus and Barbonymus gonionotus Bleeker) in two indigenous mahseer. The Tor douronensis Valenciennes were randomly collected from two locations in Sarawak (N=52), while Tor tambroides Bleeker were obtained from Peninsular Malaysia (N=56). A total of ten and twelve primers were successfully amplified producing four and five polymorphic loci in T. douronensis and T. tambroides, respectively. The number of alleles per locus ranging from 2 to 5 in T. douronensis and 2 to 7 in T. tambroides. A significant deviation from Hardy-Weinberg equilibrium (HWE) was observed at three loci (Barb37, Barb59 and Barb62) in one or more populations in T. tambroides while two loci (Barb37 and Barb62) were deviated in T. douronensis population of Batang Ai. Population structure analysis showed low level of inter-population genetic differentiation in both mahseer. Overall, the identified microsatellite loci should be useful in analysing T. douronensis and T. tambroides natural populations.

Keywords: Cross-species study; genetic characterization; mahseer; microsatellites

ABSTRAK

Kajian ini meneliti pencirian genetik dua puluh enam primer mikrosatelit yang dibentuk daripada tiga ikan siprinid (Cyprinus carpio Linnaeus, Barbus barbus Linnaeus and Barbonymus gonionotus Bleeker) ke atas dua ikan kelah indigenus. Ikan kelah Tor douronensis Valenciennes telah dipilih secara rawak dari dua tempat yang berbeza di Sarawak (N=52), manakala Tor tambroides Bleeker pula telah dikumpul secara rawak dari Semenanjung Malaysia (N=56). Sejumlah sepuluh primer berjaya diamplifikasi menghasilkan empat lokus polimorfik pada Tor douronensis; dan dua belas primer pada Tor tambroides dengan lima lokasi polimorfik. Nombor alel per lokus berjulat di antara dua hingga lima dalam Tor douronensis dan dua hingga tujuh dalam Tor tambroides. Penyimpangan daripada keseimbangan Hardy-Weinberg (HWE) yang signifikan telah dijumpai pada tiga lokus (Barb37, Barb59 dan Barb62) di dalam satu atau lebih populasi T. tambroides manakala dua lokus (Barb37 dan Barb62) mengalami penyimpangan dalam populasi T. douronensis dari Batang Ai. Analisis struktur populasi menunjukkan tahap perbezaan genetik yang rendah di peringkat inter-populasi dalam kedua-dua ikan kelah. Keseluruhannya, lokus mikrosatelit yang dikenalpasti berguna untuk menganalisis secara mendalam populasi semulajadi ikan T. douronensis dan T. tambroides.

Kata kunci: Kajian spesies-silang; kelah; mikrosatelit; pencirian genetik

INTRODUCTION

The mahseer from the genus Tor Gray such as Tor tambroides Bleeker and Tor douronensis Valenciennes, are among the most valuable and highly priced cyprinid fish in Malaysia (Litis et al. 1997). The market price for mahseer is one of the highest due to their great taste, for example, the price of T. douronensis can reach above RM100/kg while T. tambroides reaches above RM400 (USD100)/ kg in the open market in Kapit, Sarawak (Ingram et al. 2005). Thus, fishes of the genus Tor have great potential for freshwater aquaculture industry (Ingram et al. 2005). In addition, the Tor fishes are also recognized as an excellent game fish, and have high demand in the ornamental fish industry due to their attractive colourations (Ng 2004).

Therefore, realizing the economic importance of the two mahseer and given their limited distributions and population size, studies on the population structure and level of genetic variations throughout their distribution range are required for effective management and conservation strategy of this important freshwater resource. Populations of these species are declining due to degrading environmental conditions by deforestation, logging and over fishing that may have disturb their natural habitat. In India matured male and female are very difficult to find for several species of mahseer such as Tor putitora Hamilton due to over fishing by sport anglers that crave only for large fishes (Patil & Lakra 2005).

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Microsatellites or simple sequence repeats (SSRs) are short tandem repeat motifs (1-6 bases) with high levels of allelic polymorphism and co-dominant inheritance (DeWoody & Avise 2000; Jarne & Lagorda 1996; Zane et al. 2002). They are present in both coding and noncoding regions characterized by a high degree of length polymorphism (Zane et al. 2002), useful for direct assessment of pattern and distribution of genetic variability at the intraspecific level (O’Connell & Wright 1997; Primmer et al. 2006). The flanking sequences of microsatellites within related taxa are found to be highly conserved (Scribner et al. 1996) including in fish (Rico et al. 1996), allowing cross-amplification from species that diverged as long as 470 million years ago (Zane et al. 2002). Thus, the potential of developing microsatellite markers through cross-species amplification is enhanced when primers designed for one species amplify homologous loci in other species (Zardoya et al. 1996; Zheng et al. 1995). A few cross-species amplification studies had identified polymorphic microsatellite loci from other fishes useful for population genetic structure analysis of the genus Tor e.g. Tor putitora from three other cyprinids (Mohindra et al. 2004) and Tor tambroides from a catfish, Mystus nemurus (Keong et al. 2008)). The present study examines cross-species amplification of primers, developed for three cyprinids (Cyprinus carpio, Barbus barbus and Barbonymus gonionotus), in two indigenous mahseers, T. douronensis and T. tambroides. The objectives were to identify polymorphic microsatellite loci and to evaluate the suitability of the identified loci in population structure analysis of both mahseer in Malaysia.

MATERIALS AND METHODS

Mahseer samples were difficult to obtain due to their reduced numbers in most of the major rivers (Ng 2004) and their natural populations are currently confined only to the upper streams of rivers or protected areas such as national parks. Tor douronensis samples were selected randomly from two locations in Sarawak; the Batang Ai River (N=37) and the Limbang River (N=15). The Batang Ai River is a tributary of the Batang Lupar River located in the southern part of Sarawak, while the Limbang River is located in northern part of Sarawak. Meanwhile, T. tambroides samples used in this study were obtained from three locations in Peninsular Malaysia; the Sia River (N=17), the Kampung Esok River (N=20) and the Perak River (N=19). The Sia River, Pahang and the Kampung Esok River, Negeri Sembilan, both served as tributaries of the Pahang River that drained to the South China Sea while the Perak River flow west into the Straits of Malacca. The mahseer samples were morphologically identified by using the keys provided by Mohsin and Ambak (1983), Kottelat et al. (1993), and Inger and Chin (2002). Microsatellite primers from three freshwater cyprinids: Cyprinus carpio (Crooijmans et al. 1997), Barbus barbus (Chenuil et al. 1999) and Barbonymus gonionotus

(Kamonrat et al. 2002; McConnell et al. 2001) were tested for amplification of homologous loci (Table 1). The initial cross-species amplification standardization including optimization of annealing temperature for each primer pair was carried out using eight random samples of T. douronensis and T. tambroides each. The primers yielding scoreable amplified products were further evaluated using larger sample sizes to assess their suitability in the population structure analysis of both mahseer. The total DNA was extracted using the modified Cetyl Trimethylammonium Bromide (CTAB) method (Grewe et al. 1993) in the presence of Proteinase K. Polymerase chain reaction (PCR) amplifications were performed (Eppendorf) in a final volume of 10 μL, containing 25–50 ng of genomic DNA, 1X PCR buffer (10 mM Tris-HCl, pH 9.0; 50 mM KCl; 0.01% gelatin), 2.0 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer and 1.5 units of Taq DNA polymerase. Amplification conditions were 94°C for 5 min followed by 25 cycles at 94°C for 30 s, Ta for 30 seconds and 72°C for 1 min, with a final extension of 72°C for 4 min. The optimum annealing temperature (Ta), was determined through experimental standardization for each primer pair. After amplification, 10 μL of PCR products were electrophoresed on 4% high resolution MetaPhore agarose gels for 2 h at 78V/cm. The gels were stained using ethidium bromide (0.1 μL/mL) and photographed under UV light using an Alpha Imager 2200. The alleles were designated according to PCR product size and calculated relative to a standard molecular marker (20 bp and 100 bp; Cambrex). Microsatellite genetic diversity was quantified as the number of alleles (A), the allelic richness AR (the measure of the number of alleles per locus independent of the sample size), observed (Ho) and expected (He) heterozygosity values, and inbreeding coefficient (FIS or ƒ) as a measure of heterozygote deficiency or excess (Weir & Cockerham 1984) using FSTAT version 2.9.3.2 (Goudet 2001). GENEPOP version 3.3 (Raymond & Rousset 1995) was used to test genotypic distributions for conformance to Hardy–Weinberg expectations (HWE) and to test for genotypic disequilibria at each locus for each population using the Markov chain method (Guo & Thompson 1992). Genetic homogeneity tests of genotype frequency distribution at each locus were determined through an exact G- test (Goudet et al. 1996) in order to test the null hypothesis of no genetic differentiation between populations, also using FSTAT. Sequential Bonferroni adjustments (Rice 1989) were applied to correct for the effect of multiple tests. Genetic differentiation among populations was measured by the fixation index FST, calculated according to Weir and Cockerham (1984) using ARLEQUIN version 3.0 (Excoffier et al. 2005). Permutation tests (10,000 permutations) were performed in order to determine if estimates differed significantly from zero. The genetic distance between populations (rivers) in both mahseer was calculated based on an unbiased measure following

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Nei (1978). An Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) dendrogram was constructed to illustrate the relations among geographic samples using POPGENE 1.32 (Yeh & Boyle 1997). Finally, a Bayesian approach was used to infer the number of clusters (K) in the data set without prior information of the sampling locations, available in STRUCTURE version 2.0 (Pritchard et al. 2000). A model where the allele frequencies were correlated within populations was assumed (λ was set at 1, the default value). The software was run with the option of admixture, allowing for some mixed ancestry within individuals, and α was allowed to vary. Five independent runs were done for each value of K (K= 1 to 5) with a burn–in period of 25,000 iterations and 25,000 replications.

RESULTS

Of the 26 heterologous primer pairs tested, only 10 (38%) and 12 (46%) primers produced successful amplification of homologous loci in T. douronensis and T. tambroides, respectively (Table 1). In T. douronensis, four primers (Bgon13, Barb37, Barb62 and MFW7) were polymorphic exhibiting 2-5 alleles while the other six primers (Bgon22, Bgon69, Bgon75, MFW1, MFW5 and MFW11) were monomorphic when tested using all 52 individuals. In T. tambroides, five primers (Bgon13, Barb37, Barb59, Barb62 and MFW7) were polymorphic exhibiting 2-7 alleles while primers Bgon22, Bgon69, Bgon75, MFW1, MFW5, MFW11 and MFW17 produced a monomorphic band in all the 56 individuals tested. The characteristics of the polymorphic primers of both mahseer are summarized in Table 2. The observed heterozygosity (Ho) values over all loci in T. douronensis ranged from 0.0606 (Locus MFW7 of Batang Ai) to 0.3939 (locus Barb62 of Batang Ai) (Table 4). In T. tambroides, the Ho values ranged from 0.0000 (locus MFW7 of N. Sembilan) to 0.8421 (locus Barb59 of Perak) (Table 3). Test of linkage disequilibrium between loci found no significant disequilibrium among pairwise comparisons (data not shown). A significant deviation from Hardy-Weinberg equilibrium (HWE) was observed at three loci (Barb37, Barb59 and Barb62) in one or more populations in

T. tambroides (Table 3) while two loci (Barb37 and Barb62) deviated in the T. douronensis population of Batang Ai (Table 4). Homogeneity test found significant heterogeneity (p <0.05) in genotype proportions after Bonferonni correction observed at locus Barb37 in T. douronensis while three of five loci (MFW7, Barb59 and Barb62) were significant in T. tambroides. Combined probabilities over all loci were significant (p <0.05) (data not shown). Pairwise estimates of FST over all loci between samples in both species are presented in Table 5. Within T. tambroides, only one (between the Pahang and the Perak populations) pairwise estimate of FST showed significant genetic differentiation (p< 0.05) while pairwise estimates of FST showed no significant differentiation between T. douronensis populations from Batang Ai and Ulu Limbang. Pairwise estimates of genetic distances computed by Nei (1978) among populations (Table 5) showed that the highest genetic distance was between T. tambroides population from Pahang and T. douronensis population from Ulu Limbang (FST=0.4187). Bayesian cluster analysis performed with STRUCTURE showed that the most likely K value identified was K = 2, and results from other K values (K= 3 to K= 5) did not identify any formation of additional clusters (Figure 1). The two identified cluster correspond to the two mahseer studied; Cluster 1 consisted of the three T. tambroides populations while Cluster 2 consisted of the two T. douronensis populations. No evidence of population substructuring was found in either species based on the STRUCTURE analysis. The UPGMA dendrogram also generated two clusters corresponding to the two species studied (Figure 2), similar to those identified by STRUCTURE.

DISCUSSION

The results of this study showed the potential of finding polymorphic microsatellites loci through a rapid non-cloning method. Although only a small proportion of polymorphic loci (10% (four out of 26) in T. douronensis and 12% (five out of 26 loci) in T. tambroides) were found, the genetic diversity parameters were comparable to the results found in other cross-species amplification studies of mahseer. This includes a study by Keong et al. (2008)

TABLE 1. Primers of microsatellite loci tested for cross-species amplification in Tor douronensis and Tor tambroides

Source species Number of primer pairs tested

Locus References Successful amplification (n(%))T. douronensis T. tambroides

Barbonymus gonionotus

10 Bgon2, 8, 12, 13, 17, 19, 22, 69, 75, 79

McConnell et al. 2001, Kamonrat et al. 2002

4 (40) 4(40)

Barbus barbus 4 Barb37, 54, 59, 62 Chenuil et al. 1999 2 (50) 3(75)Cyprinus carpio 12 MFW1, 2, 5, 7, 11, 15,

17, 18, 19, 24, 26, 28Crooijmans et al. 1997 4 (33) 5(42)

Total tested 26 10 (38) 12(46)

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TABLE 2. Characteristics of amplified microsatellite loci in details of Tor douronensis and T. tambroides

Resource species T. douronensis T. tambroides

Species Locus Primer sequence (5’ to 3’) Repeat motif

Ta (ºC) Ta (ºC)

No of alleles

Ta (ºC) No of alleles

Barbonymus gonionotus

Bgon13

Bgon22

Bgon69

Bgon75

F: CCCGTGCAATTCAATATGR: TAAGTAGCACAGATGTGAGGF: TCTTGTTGATCACACGGACGR: GTGACTGTATCAATGAGTCTGF: GCAAAGGTTCTGTCAAGGR:GTATCCAGAAACATGTTCAGF: CTGGTAAAGACTTCAGATGCR: GCATGCAAAATGAGAAAGGCT

GT

TCC

TG

AC

53

49

49

53

50

50

46

46

2

2

1

1

50

50

46

46

2

2

1

2

Barbus barbus

Barb37

Barb59

Barb62

F: AAATACGCTCTCCTCATTACR: GTACAAAAGCAAAAATAAATTAF: CTGTATCCATCACATAGGCTR: CATGATTTAATAGAACACACACF: GGCACAAAAATGGATTCATATCR: GTACACGAGCATATGGACAA

ATTT

GATA

ATTT

50

56

58

50

-

50

3

-

5

50

50

50

3

7

4

Cyprinus carpio

MFW1

MFW5

MFW7

MFW11

MFW17

F: GTCCAGATCGTTCATCAGGAGR: GAGGTGTACACTGAGTCACGCF: GAGATGCCTGGGGAAGTCACR: AAAGAGAGCGGGGTAAAGGAGF: TACTTTGCTCAGGACGGATGCR: ATCACCTGCACATGGCCACTGF: GCATTTGCCTTGATGGTTGTGR: TCGTCTGGTTTAGAGTGCTGCF: CAACTACAGAGAAATTTCATGR: GAAATGGTACATGACCTCAAG

CA

CA

CA

CA

CA

55

55

55

55

55

50

65

65

50

-

1

1

2

1

-

50

65

65

50

50

1

1

2

1

1

TABLE 3. Parameters of genetic variability for polymorphic microsatellite locus in T. tambroides samples from three rivers. Given are number of alleles (A), allelic richness (AR), inbreeding coefficient (FIS), observed (Ho) and expected (He)

heterozygosity values, the probability of Hardy-Weinberg equilibrium (HWE) and the probability of genotype homogeneity between samples

Locus Population A AR Ho He FISHWE

(p-value)1Genotype homogeneity

(p-value)2

MFW7 Pahang N. Sembilan Perak

2 1 2

2.0000 1.0000 2.0000

0.3529 0.0000 0.3158

0.2907 0.0000 0.2659

-0.2143 0.0000

-0.1875

0.4256 -

0.4606

0.0100*

Barb37 PahangN. SembilanPerak

333

3.00002.80003.0000

0.23530.55000.2632

0.47920.47120.4806

0.5090-0.16710.4525

0.0011**

0.43710.0000*

0.3620

Barb59 PahangN. SembilanPerak

747

7.00003.79406.526

1.00000.50000.8421

0.68160.57750.7604

-0.46700.1342

-0.1705

0.0009*

0.0278*

0.0003*

0.0000**

Barb62 PahangN. SembilanPerak

343

3.00003.60002.9790

0.52940.15000.5263

0.48620.33620.4598

0.15300.5539

-0.1446

0.0001*

0.0134*

0.0000**

0.0000**

Bgon13 PahangN. SembilanPerak

222

1.99801.96401.9980

0.42350.10000.1579

0.11070.09500.1454

-0.0625-0.0526-0.0857

0.85750.86940.7632

0.8100

(P-value)1**P<0.001 and **P<0.01(P-value)2**P<0.001

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TABLE 4. Parameters of genetic variability for polymorphic microsatellite locus in T. douronensis samples from three rivers. Given are number of alleles (A), allelic richness (AR), inbreeding coefficient (FIS), observed (Ho) and expected (He) heterozygosity values,

the probability of Hardy-Weinberg equilibrium (HWE) and the probability of genotype homogeneity between samples

Locus Population A AR Ho He FISHWE

(p-value)1Genotype

homogeneity (p-value)2

MFW7 Batang AiUlu Limbang

22

1.63602.0000

0.06060.0769

0.05880.0740

-0.0313-0.0400

0.89971.0000

0.5850

Barb37 Batang AiUlu Limbang

32

2.97902.0000

0.24240.0769

0.41550.0740

0.4166-0.0400

0.0000**

1.00000.0180*

Barb62 Batang AiUlu Limbang

43

3.38103.0000

0.39390.3846

0.60280.5178

0.34650.2571

0.0012**

0.51530.1980

Bgon13 Batang AiUlu Limbang

22

1.92602.0000

0.15150.0769

0.14000.0740

-0.0820-0.0400

0.67581.0000

0.3640

(P-value)1**P<0.001 and **P<0.01(P-value)2**P<0.001

TABLE 5. Estimates of pairwise genetic distances (Nei 1978; below diagonal) and FST (Weir & Cockerham 1984; upper diagonal) among populations of T. tambroides and T. douronensis

T. tambroides T. douronensis

Pahang N. Sembilan Perak Batang Ai Ulu Limbang

T. ta

mbr

oide

s Pahang - 0.0409 0.0499* 0.3170* 0.4187*

N. Sembilan 0.0134 - 0.0000 0.2238* 0.2724*

Perak 0.0181 0.0000 - 0.2163* 0.2581*

T. d

ouro

nens

is Batang Ai 0.1952 0.1490 0.1412 - 0.0361

Ulu Limbang 0.2201 0.1526 0.1416 0.0128 -

*P-value, significant level (P< 0.05)

in T. tambroides using five polymorphic microsatellites developed from a catfish (Mystus nemurus) (number of allele per locus ranged from 1 to 7 and observed heterozygosities ranged from 0.2400 to 1.0000), and a study by Mohindra et al. (2004) in Tor putitora using seven (22%) polymorphic (out of a total of 32 primers tested) microsatellites developed from Catla catla, C. carpio and B. barbus (number of alleles per locus ranged from 1 to 10 and observed heterozygosities ranged from 0.0000 to 0.9000). However, the current genetic diversity results were lower than those found by Nguyen et al. (2006) in microsatellites developed for T. tambroides (number of alleles per locus ranged from 1 to 9 and observed heterozygosities ranged from 0.0390 to 0.7510) and subsequent cross-species study in T. douronensis by

Nguyen (2007) (number of alleles per locus ranged from five to 21 and observed heterozygosities ranged from 0.0400 to 0.7850). Nevertheless, the results of this study supported the hypothesis that certain sequences flanking the microsatellite regions of the genome might be conserved among the cyprinids (Zane et al. 2002), thus potentially allowing primers to be used interspecifically among cyprinids (Lal et al. 2004; Mohindra et al. 2004; Nguyen 2007; Yue & Orban 2002 ). The results of this study also showed that the optimum annealing temperature (Ta ºC) observed in both mahseer differed from that reported in the source species for the respective primer pair, except in Barb37 similar to the findings by Mohindra et al. (2004). The fact that eight out of 12 (67%) of the successfully amplified primers in this study exhibited annealing temperature lower that

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those found in the source species supported the general assumption that cross-species amplification tends to have lower annealing temperature as compared with the species where the primer(s) were originally developed (Zane et al. 2002). Two and three out of the five polymorphic loci were not in Hardy-Weinberg equilibrium (HWE) in T. douronensis and T. tambroides in one or more population, respectively. Departure from HWE may result from one or more of the followings reasons: (i) Sampling error because only a small sample size was studied, thus did not have a true representation of the population allele frequencies (Mohindra et al. 2004). (ii) Wahlund effect, i.e. presence of fewer heterozygotes in a population than predicted on account of a population subdivision (Kumar et al. 2006). (iii) presence of null alleles as suggested by excess of homozygotes for most allele size classes (Nguyen 2007) and (iv) Reduction in the effective breeding population size in T. tambroides as a result of overexploitation

and/or anthropogenic disturbances (i.e. river pollution, deforestration, watershed erosion etc). Our microsatellite analysis showed low levels of genetic differentiation among the three T. tambroides populations of Peninsular Malaysia (N. Sembilan, Pahang and Perak) similar to the results found using mitochondrial cytochrome c oxidase I sequences (Esa et al. 2008). In addition, the two T. douronensis populations (Batang Ai and Ulu Limbang) separated by high geographical distance (around 800 km) also showed very low genetic substructuring between them and was not concordant with the high population structuring results found by Nguyen (2008). Thus, the inclusion of more polymorphic microsatellite loci and of sample sizes in each population might provide a better resolution of the population genetic structure of the two mahseer species. Overall, the cross-species amplification study identifies five polymorphic microsatellite loci with considerable genetic variation potentially useful in the fine

FIGURE 1. Proportional membership (Q) of each individual of T. tambroides and T. douronensis identified by STRUCTURE at K=2 to K=5. The numbers in theX-axis correspond to a specific population:

1-Negeri Sembilan, 2-Pahang, 3-Perak, 4-Batang Ai, 5-Ulu Limbang

1.000.80

0.60

0.40

0.20

0.00

1.000.80

0.60

0.40

0.20

0.00

1.000.80

0.60

0.40

0.20

0.00

1.000.800.600.400.200.00

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scale population structure analysis of T. douronensis and T. tambroides natural populations.

ACKNOWLEDGEMENTS

The authors would like to thank Dr Stephen Sungan and staffs of the IFPRC, Tarat, Sarawak, the Fisheries Department of Malaysia and Mr. M. F. Yusof from UPM for their assistances. This project was funded by the National Biotechnology Directorate (MOSTI) grant no: 01-02-04-008/BTK/ER/33.

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FIGURE 2. UPGMA dendrogram showing the genetic relationships among populations of T. tambroides and T. douronensis

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Yuzine B. EsaDepartment of ZoologyFaculty of Resource Science and TechnologyUniversiti Malaysia Sarawak 94300 Kota Samarahan, Sarawak, Malaysia

Khairul Adha A. RahimDepartment of Aquatic SciencesFaculty of Resource Science and Technology Universiti Malaysia Sarawak 94300 Kota Samarahan, Sarawak, Malaysia

Siti Shapor Siraj* & Muhammad Fadhil Syukri Department of AquacultureFaculty of AgricultureUniversiti Putra Malaysia 43400 Serdang Selangor D.E.Malaysia

Siti Khalijah Daud & Ho Ghim ChongBiology Department

1095

Faculty of ScienceUniversiti Putra Malaysia 43400 Serdang, Selangor D.E.Malaysia

Tan Soon Guan

Department of Cell and Molecular BiologyFaculty of Biotechnology and Biomolecular SciencesUniversiti Putra Malaysia 43400 Serdang Selangor D.E.Malaysia

*Corresponding author; email: shaporsiraj@yahoo.com

Received: 23 August 2010Accepted: 14 December 2010